The present invention, in some embodiments thereof, relates to methods of ex vivo differentiating mesenchymal stem cells towards neural stem cells and motor neurons using microRNAs (miRNAs).
Mesenchymal stem cells (MSCs) are a heterogeneous population of stromal cells that can be isolated from multiple species, residing in most connective tissues including bone marrow, adipose, placenta, umbilical cord and perivascular tissues. MSCs can also be isolated from the placenta and cord's Wharton's jelly.
The concentration of MSCs in all tissues, including bone marrow and adipose tissue is very low but their number can be expanded in vitro. Typically, expansion of MSCs using up to 15 passages does not result in mutations indicating genetic stability. MSC can differentiate into cells of the mesenchymal lineage, such as bone, cartilage and fat but, under certain conditions, have been reported to acquire the phenotype of cells of the endodermal and neuroectodermal lineage, suggesting some potential for “trans differentiation”.
Within the bone marrow compartment, these cells are tightly intermingled with and support hematopoiesis and the survival of hematopoietic stem cells in acquiescent state (7). In addition, after expansion in culture, MSCs retain their ability to modulate innate and adaptive immunity (8). Furthermore, MSCs migrate actively to sites of inflammation and protect damaged tissues, including the CNS, properties that supported their use as new immunosuppressive or rather immunoregulatory or anti-inflammatory agents for the treatment of inflammatory and immune-mediated diseases including autoimmune disorders (9). These features of MSCs merited their use to control life-threatening graft-versus-host-disease (GVHD) following allogeneic bone marrow transplantation, thus controlling one of the most serious complications of allogeneic bone marrow transplantation, helping to lower transplant-related toxicity and mortality associated with multi-system organ injury (10).
Several studies have shown that MSCs following exposure to different factors in vitro, change their phenotype and demonstrate neuronal and glial markers [Kopen, G. C., et al., Proc Natl Acad USA. 96(19):10711-6, 1999; Sanchez-Ramos, et al. Exp Neurol. 164(2):247-56. 2000; Woodbury, D., J Neurosci Res. 61(4):364-70, 2000; Woodbury, D., et al., J Neurosci Res. 69(6):908-17, 2002; Black, I. B., Woodbury, D. Blood Cells Mol Dis. 27(3):632-6, 2001; Kohyama, J., et al. Differentiation. 68(4-5):235-44, 2001; Levy, Y. S. J Mol Neurosci. 21(2):121-32, 2003].
Accordingly, MSCs (both ex-vivo differentiated and non-differentiated) have been proposed as candidates for cell replacement therapy for the treatment of various neurological disorders including multiple sclerosis, Parkinson's disease, ALS, Alzheimer's disease, spinal cord injury and stroke.
Motor neurons in the spinal cord innervate skeletal muscles, and originate from neuroepithelial cells in a restricted area of the developing spinal cord (neural tube). During embryonic development, motor neurons extend their processes (nerves) to the periphery to innervate skeletal muscles that are adjacent to the spinal cord. In an adult human body, however, motor neuron's axons are projected large distances away from the cell bodies in the spinal cord to reach their target muscles. Because of this, motor neurons have a higher metabolic rate compared to smaller neurons, and this renders them more susceptible to genetic, epigenetic, and environmental changes. Motor neurons can not renew themselves and therefore their loss or degeneration are generally associated with fatal neurological conditions including paralysis and disorders such as pediatric spinal muscular atrophy (SMA) and adult onset amyotrophic lateral sclerosis (ALS).
Roy et al., 2005 [Exp Neurol. 2005; 196:224-234]; Zhang et al., 2006 [Stem Cells. 2006; 24:434-442]; Bohl et al., 2008 [Stem Cells. 2008; 26:2564-2575]; and Dimos et al., 2008 [Science. 2008; 321:1218-1221] the contents of which are incorporated by reference teach genetic modification of different stem cells to induce differentiation into motor neurons.